Synthesis of nucleoside mono- and triphosphates bearing oligopyridine ligands, their incorporation into DNA and complexation with transition metals

Abstract

Modified nucleoside mono- (dA^RMPs and dC^RMPs) and triphosphates (dA^RTPs and dC^RTPs) bearing bipyridine or terpyridine ligands attached viaacetylene linker were prepared by single-step aqueous-phase Sonogashira cross-coupling of 7-iodo-7-deaza-dAMP or -dATP, and 5-iodo-dCMP or -dCTP with the corresponding bipyridine- or terpyridine-linked acetylenes. The modified dN^RTPs were successfully incorporated into the oligonucleotides by primer extension experiment (PEX) using different DNA polymerases and the PEX products were used for post-synthetic complexation with Fe²⁺.

---

Introduction

Nucleic acids bearing diverse functional groups are attracting growing interest due to their potential applications in chemical biology, bioanalysis, or nanotechnology.¹ Transition metal complexes within (metal-base-pairs² or intercalators³) and outside (metal complex covalently attached to a nucleobase, sugar or phosphate)^4–7 the DNA duplex have been extensively studied. Diverse oligopyridine-metal complexes attached to DNA have been prepared and applied in stabilization of duplexes,⁴redox⁵ and photoredox⁶labeling or for self assembly of 2D and 3D nanostructures.⁷ We^8,9 and others¹⁰ have previously developed efficient syntheses of bipyridine- (bpy) and terpyridine- (tpy) modified nucleoside building blocks. However, despite the apparent application potential, chemical synthesis of oligopyridine-nucleobase linked oligonucleotides (ONs) and their post-synthetic complexations have been reported very scarcely.¹¹ In parallel with our project, the Wagenknecht group has developed¹² a chemical synthesis of tpy-linked ONs and their complexation with Ni²⁺ and Zn²⁺.

Apart from chemical synthesis,¹³ base-modified DNA can be also prepared enzymatically^14,15 by polymerase incorporation of base- modified 2′-deoxyribonucleoside triphosphates (dNTPs) bearing substituents at position 5 of pyrimidines or at position 7 of 7-deazapurines. Combination of a direct cross-coupling modification of dNTPs with enzymatic incorporation resulted in efficient and general two-step synthesis of base-modified DNA.¹⁶ Here we report the synthesis of oligopyridine-linked nucleotides and dNTPs and enzymatic synthesis and complexation of oligopyridine-linked DNA.

Result and discussion

Synthesis of modified nucleotides and dNTPs

In order to prepare modified oligonucleotidesvia enzymatic incorporation, oligopyridine ligands had to be attached to the nucleobase in dNTPs via suitable linker (in our case rigid, linear and electron-conjugate acetylene tether). To develop the synthetic methodology and complexation of ONs, the chemistry was first performed on model nucleosides monophosphates (dNMPs). The first target compounds of our choice were 7-substituted 7-deaza-2′-deoxyadenosine (dA^RMPs) and 5-substituted 2′-deoxycytidine 5′-monophosphates (dC^RMPs). They were prepared by single-step aqueous-phase Sonogashira cross-coupling reaction of halogenated nucleoside monophosphates dA^IMP and dC^IMP with terminal acetylenes 1a–c¹⁸ (Scheme 1, Table 1). The reactions were generally performed in the presence of Pd(OAc)₂, water soluble tris(3-sulfonatophenyl)phosphane (TPPTS) ligand, CuI and Hünnig base in the mixture water/acetonitrile (2 : 1) at 80 °C for 1.5 h. The desired corresponding products dA^6bpyMP, dA^5bpyMP, dA^tpyMP and dC^6bpyMP, dC^5bpyMP, dC^tpyMP were isolated after the purification on reverse-phase HPLC in good yields (from 47% to 89%, Table 1).

Table 1 Synthesis of modified dN^RMPs by Sonogashira cross-coupling

Entry	Monophosphate	Alkyne	Product	Yield
1	dC ^I MP	1a	dC ^6bpy MP	89%
2	dC ^I MP	1b	dC ^5bpy MP	89%
3	dC ^I MP	1c	dC ^tpy MP	47%
4	dA ^I MP	1a	dA ^6bpy MP	52%
5	dA ^I MP	1b	dA ^5bpy MP	70%
6	dA ^I MP	1c	dA ^tpy MP	57%

---

Scheme 1 Synthesis of modified dN^RMPs and dN^RTPs. Reagents and conditions: i) Pd(OAc)₂ (5 mol%), TPPTS (5 equiv. to Pd), CuI (10 mol%), Et(i-Pr)₂N (10 equiv.), H₂O/CH₃CN (2 : 1), 80 °C.

Having developed the methodology for modification of nucleotides, we have proceeded with the direct functionalization of dNTPs by Sonogashira cross-coupling, in analogy to our previously developed procedures^5,16 (Scheme 1, Table 2). To avoid the hydrolysis of the starting and final triphosphates, the reaction mixture was heated at 80 °C only for 1 h.^5,16 The corresponding products dA^6bpyTP, dA^5bpyTP, and dA^tpyTP, as well as dC^6bpyTP, dC^5bpyTP, and dC^tpyTP were isolated after the purification on reverse-phase HPLC in good yields (40–48% for dA^RTPs and 59–69% for dC^RTPs, Table 2).

Table 2 Synthesis of modified dN^RTPs by Sonogashira cross-coupling

Entry	Triphosphate	Alkyne	Product	Yield
1	dC ^I TP	1a	dC ^6bpy TP	67%
2	dC ^I TP	1b	dC ^5bpy TP	59%
3	dC ^I TP	1c	dC ^tpy TP	69%
4	dA ^I TP	1a	dA ^6bpy TP	42%
5	dA ^I TP	1b	dA ^5bpy TP	48%
6	dA ^I TP	1c	dA ^tpy TP	40%

---

Incorporation of modified dNTPs by DNA polymerases

All the functionalized dA^RTPs or dC^RTPs were tested as substrates for several thermostable DNA polymerases in primer extension experiment (PEX). Each PEX experiment, analyzed by denaturing polyacrylamide gel electrophoresis (PAGE), was compared with positive (all four natural dNTPs) and negative control experiments (absence of one natural dNTPs) in order to exclude any mis-incorporations. Single and multiple incorporation of oligopyridine-functionalized triphosphate were tested (for sequences of primer and templates see Table 3).

Table 3 Primer and templates used for primer extension^a

a In the template (temp) ONs segments that form a duplex with primer are printed in italics, the replicated segments are printed in bold. For magnetic separation of the extended primer strands, the templates 5′-end biotinylated. The acronyms used in the text for primer products are analogs to those introduced for templates (e.g. the PEX product pex^rnd16 was synthesized on temp^rnd16 template.
prim^rnd	5′-CATGGGCGGCATGGG-3′
temp^rnd16	5′-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3′
temp^C	5′-CCCGCCCATGCCGCCCATG-3′
temp^A	5′-CCCTCCCATGCCGCCCATG-3′
temp^A1	5′-TCCCATGCCGCCCATG-3′

---

Single-nucleotide extension experiments were tested separately with each of the three dA^RTPs and dC^RTPs by using 19-mer templates temp^A and temp^C (Fig. 1). Experiments using Pwo polymerase were mostly successful to give fully extended products for all of the dNTPs except dC^tpyTP (lane 11), which gave the mixture of fully and partially extended ONs. Therefore the reaction condition for incorporation of dC^RTPs were further optimized and the use of DyNAzyme II enzyme leads to fully extended products. The lack of extension in negative control (A- or C-) proves that no miss-incorporation occurred. Each spot of a PEX-product (even in the positive and negative control experiments) is accompanied by a weak band of one nucleotide shorter product due to 3′-5′ exonuclease activity of the enzyme.

Fig. 1 Denaturing PAGE analysis of PEX experiment synthetized on temp^A (lines 2–6) and temp^C (lines 7–16) with Pwo or DyNAzyme II polymerases. 5′-³²P-end labelled primer-template was incubated with different combinations of natural and functionalized dNTPs. P: Primer; A+: natural dATP, dGTP; A-: dGTP; A^6bpy: dA^6bpyTP, dGTP; A^5bpy: dA^5bpyTP, dGTP; A^tpy: dA^tpyTP, dGTP; C+: natural dCTP, dGTP; C-: dGTP; C^6bpy: dC^6bpyTP, dGTP; C^5bpy: dC^5bpyTP, dGTP; C^tpy: dC^tpyTP, dGTP.

In order to compare the efficiency in incorporation of the oligopyridine-modified dNTPs in comparison with the natural ones, we have performed a simple kinetics study in single-nucleotide PEX reaction. The experiments with unmodified and modified dATPs were performed using Pwo polymerase and two modified dA^RTPs in comparison with the natural dATP (Fig. 2). The PEX with the natural dATP was finished within 1 min whereas the PEX with dA^5bpyTP took 2 min and, with the more bulky dA^tpyTP, even 5 min to complete. This shows that the modified dA^RTPs are worse substrates for the polymerase. Therefore, the reaction time for multiple incorporations must be prolonged to 30 min to ensure full length product formation.

Fig. 2 Comparison of the rate of the single-nucleotide PEX with natural A+ (dATP) and modified (dA^5bpyTP, dA^tpyTP) nucleotides using Pwo polymerase with temp^A1 without natural dGTP. The reaction mixtures were incubated for time intervals indicated (in minutes), followed by stopping the reaction by addition of PAGE loading buffer and immediate heating.

Multiple incorporations were tested on 31-mer template temp^rnd16 containing four copies of each of the four bases and requiring incorporation of four modified dN^RTPs in separate positions. Several polymerases were tested: Pwo, DyNAzyme II, Vent (exo⁻), Deep Vent, Deep Vent (exo⁻), Phusion, KOD XL and Therminator. Incorporation of dA^RTPs using Pwo or Deep Vent leads to fully extended ONs (Fig. 3, lanes 5–7), while incorporation of dC^6bpyTP and dC^tpyTP (lanes 8 and 10) gave a mixture of ONs of different length (for incorporation using Deep Vent and other polymerasess see ESI†). Even after the optimization of condition (testing different DNA polymerases, higher concentration of enzyme and dC^RTPs) the incorporation of dC^tpyTP was less feasible and resulted in early termination of PEX (lane 15). Five time higher concentration of DyNAzyme II DNA polymerase in combination with double concentration of dC^RTP was the most efficient in the incorporation of dC^RTP. The products of pex^rnd16 slightly differ in electrophoretic mobilities visible on gel partly (combination of the effects of the higher molecular weight and possible secondary structure formation). Therefore, the successful incorporation and full-length products were verified by measurement of MALDI mass spectra of PEX products. Single stranded DNA was prepared by PEX with biotinylated templates and then isolated by magnetoseparation.^16b The correct masses were confirmed for the fully-extended products (see ESI†).

Fig. 3 Denaturing PAGE analysis of PEX experiment synthesized on temp^rnd16 with Pwo and DyNAzyme II polymerases. 5′-³²P-end labelled primer-template was incubated with different combinations of natural and functionalized dNTPs. P: Primer; +: natural dNTPs; A-: dTTP, dCTP, dGTP; C-:dATP, dTTP, dGTP; A^6bpy: dA^6bpyTP, dTTP, dCTP, dGTP; A^5bpy: dA^5bpyTP, dTTP, dCTP, dGTP; A^tpy: dA^tpyTP, dTTP, dCTP, dGTP; C^6bpy: dC^6bpyTP, dATP, dTTP, dGTP; C^5bpy: dC^5bpyTP, dATP, dTTP, dGTP; C^tpy: dC^tpyTP, dATP, dTTP, dGTP.

Complexation studies

All six nucleoside monophosphates (dN^RMPs) were tested as model compounds for further complexation studies on oligopyridine modified oligonucleotides. Aqueous solutions of dN^RMPs were mixed with 0.5 equiv. of divalent metal such as Cu²⁺, Zn²⁺, Ni²⁺ and Fe²⁺. After incubation for 10 min. at room temperature, the complex formation was detected by UV/Vis spectroscopy. The spectra were recorded for the non-metalated and metalated monophospate. While MLCT bands of bpy- or tpy-modified monophosphates with Cu²⁺, Zn²⁺, Ni²⁺ are partially overlapping with dominating absorbance at 350 nm due to bpy and tpy ligands (see ESI†), the complex formed by mixingdN^tpyMP with Fe²⁺ can be easily detected due to characteristic absorbance at 580 nm¹⁷ (Fig. 4, magenta line).

Fig. 4 UV/Vis spectra of: A) dA^tpyMP, B) dC^tpyMP with divalent metals.

After successful complexation of nucleoside monophosphates, we proceeded with complexation of tpy-modified oligonucleotides with Fe²⁺, due to this easy detectable MLCT band. For the complexation studies detected by UV/Vis spectroscopy, we have chosen ON prepared by PEX on larger scale using Deep Vent polymerase and template temp^rnd16, due to the higher concentration of tpy-modification in product. Natural DNA was prepared by incorporaion of dCTP, dGTP, dTTP and dATP, while the modified ON was prepared by using dA^tpyTP as surrogates of natural dATP. Oligonucleotides had to be purified to remove unincorporated dA^tpyTP. After addition of 0.5 equiv. of Fe(BF₄)₂·6H₂O per each modification and incubation for several hours at room temperature, the UV/Vis spectra were recorded for non-metalated and metalated DNA duplexes, either natural or modified one (Fig. 5). The dominant absorbance at ca. 260 nm originates from the absorbance of natural nucleotides (black line), small absorbance band at ca. 350 nm is due to the presence of tpy-modification (green line) while the band at ca. 590 is the MLCT band^7c of complex formed by tpy-modified ON with Fe²⁺ (blue line). Similar MLCT band is not observed for the natural DNA mixed with Fe²⁺ (red line).

Fig. 5 UV/Vis spectra of natural and modified DNA duplexes with Fe(BF₄)₂·6H₂O.

The complex formation was also detected by native polyacrylamide gel electrophoresis. For the first experiments (monoincorporations), ONs were prepared by the PEX experiment using temp^A and dATP (natural DNA) or dA^tpyTP, or temp^C and dCTPs (natural DNA) or dC^tpyTP. The PEX products were directly, without previous purification, mixed with 1 eqiv. of Fe(BF₄)₂·6H₂O or FeCl₂ (calculated to the amount of modified dN^tpyTP in PEX experiment) and incubated at room temperature for 3 h. Successful complex formation of pex^A and pex^C, containing one tpy-modification with Fe²⁺ ions is clearly shown by bands with slower mobility (Fig. 6, lanes 4 and 8), while no change in mobility was not observed for natural DNA mixed with Fe²⁺ ions (lanes 2 and 6).

Fig. 6 Non-denaturing gel electrophoresis (8% SB_PAGE) of DNA duplexes in the absence and in the presence of Fe²⁺ for pex^A (A) or pex^C (B). 5′-³²P-end labelled primer-template was incubated with different combinations of natural and functionalized dNTPs: A+: unmodified DNA (dATP, dGTP); A+/Fe²⁺: unmodified DNA (dATP, dGTP) mixed with Fe²⁺; A^tpy: tpy-modified DNA (dA^tpyTP, dGTP); A^tpy/Fe²⁺: tpy-modified DNA (dA^tpyTP, dGTP) mixed with Fe²⁺; C+: unmodified DNA (dCTP, dGTP); C+/Fe²⁺: unmodified DNA (dCTP, dGTP) mixed with Fe²⁺; C^tpy: tpy-modified DNA (dC^tpyTP, dGTP); C^tpy/Fe²⁺: tpy-modified DNA (dC^tpyTP, dGTP) mixed with Fe²⁺.

Similar results were shown also for pex^rnd16 containing four tpy-modifications (Fig. 7). Since the dC^tpyTP was not a good substrate in multiple incorporations, this experiment was only performed with A^tpy. Here the bands of the complexes are more smeared since mixtures of possible products are formed.

Fig. 7 Non-denaturing gel electrophoresis (8% SB_PAGE) of DNA duplexes in the absence and in the presence of Fe²⁺ pex^rnd16. 5′-³²P-end labelled primer-template was incubated with different combinations of natural and functionalized dNTPs: A+: unmodified DNA (dATP, dTTP, dCTP, dGTP); A+/Fe²⁺: unmodified DNA (dATP, dTTP, dCTP, dGTP) mixed with Fe²⁺; A^tpy: tpy-modified DNA (dA^tpyTP, dTTP, dCTP, dGTP); A^tpy/Fe²⁺: tpy-modified DNA (dA^tpyTP, dTTP, dCTP, dGTP) mixed with Fe²⁺.

Conclusions

Novel dN^RMPs and dN^RTPs bearing oligopyridine ligands attached viaacetylene linker were prepared in single-step aqueous-phase Sonogashira cross-coupling reaction of iodinated dN^IMPs or dN^ITPs with corresponding terminal acetylene. Functionalized dN^RTPs were shown to be good substrates for DNA polymerases and were incorporated into the DNA by primer extension. Pwo polymerase, which was successfully used for incorporation of modified dA^RTPs, did not incorporate dC^RTPs with the same efficiency. For incorporation of modified dC^RTPs, DyNAzyme II was identified as the most suitable enzyme, although the multiple incorporation of dC^tpyTP was still less successful, resulting early termination of PEX experiment. Oligopyridine functionalized ONs, containing either one or four modifications, were successfully used for post-synthetic complexation with Fe²⁺ metals ions. Therefore, the A^tpy-containing DNA has potential for self-assembly studies.

Experimental

Sonogashira cross-coupling reaction were performed under argon atmosphere. Oligopyridinyl acetylenes,¹⁸ halogenated monophosphates^16g,19 and halogenated triphosphates^16a,c were prepared according to the literature procedures. Other chemicals were purchased from commercial suppliers and were used as received. Preparative HPLC separations were performed on column packed with 10 μm C18 reversed phase (Phenomenex, Luna C18(2)). NMR spectra were measured on a Bruker 500 or Bruker 600 (500 or 600 MHz for ¹H, 125.7 or 150.9 MHz for ¹³C and 202.3 for ³¹P) in D₂O (referenced to dioxane as internal standard, _δH = 3.75 ppm, _δC = 69.3 ppm, standard for ³¹P NMR was external H₃PO₄) or in CD₃OD (referenced to TMS as an internal standard). Chemical shifts are given in ppm (δ scale), coupling constants (J) in Hz. Complete assignment of all NMR signals was achieved by use of a combination of H,H-COSY, H,C-HSQC, and H,C-HMBC experiments. NMR spectra of dNTPs were measured in phosphate buffer at pH 7.1. Mass spectra were measured on LCQ classic (Thermo-Finnigan) spectrometer using ESI or Q-Tof Micro (Waters, ESI source, internal calibration with lockspray). Mass spectra of functionalized DNA were measured by Maldi-TOF, Reflex IV (Bruker) with nitrogen laser. UV/Vis spectra were measured on Varian CARY 100 Bio spectrophotometer at room temperature.

General procedure for Sonogashira cross-coupling – synthesis of modified dN^RMPs

Mixture CH₃CN/H₂O (1 : 2) (1.5 ml) and Et(i-Pr)₂N (10 equiv.) were added to an argon-purged flask containing halogenated nucleoside monophosphatedC^IMP or dA^IMP (60 mg), an alkyne 1a–c (1.5 equiv.) and CuI (10 mol%). In a separate flask, Pd(OAc)₂ (5 mol%) and TPPTS (5 equiv. to Pd) were combined, evacuated and purged with argon followed by addition of CH₃CN/H₂O (1 : 2) (0.5 ml). The mixture of catalyst was then injected into the reaction mixture and the reaction mixture was stirred at 80 °C for 1.5 h. The solvent was evaporated in vacuo. Products were purified by semi-preparative HPLC on C18 column using linear gradient of 0.1 M TEAB (triethylamonium bicarbonate) in H₂O to 0.1 M TEAB in H₂O/MeOH (1 : 1) as an eluent. Several co-distillations with water and conversion to sodium salt form (Dowex 50 in Na⁺ cycle) followed by freeze-drying from water, gave the products as brownish or yellowish powder.

5-[(2′′,2′′′-bipyridin-6′′-yl)ethynyl]-2′-deoxycytidine 5′-O-monophosphate (dC^6bpyMP)

This compound was prepared according to the general procedure from 5-iodo-2′-deoxycytidine monophosphate dC^IMP and 1a in the yield 89%.

¹H NMR (600.1 MHz, CD₃OD): 2.26 (ddd, 1H, J_gem = 13.7, J_2′b,1′ = 7.1, J_2′b,3′ = 6.2, H-2′b); 2.41 (ddd, 1H, J_gem = 13.7, J_2′a,1′ = 6.0, J_2′a,3′ = 3.6, H-2′a); 4.05 (dt, 1H, J_gem = 10.4, J_H,P = J_5′b,4′ = 4.4, H-5′b); 4.10 (m, 1H, H-4′); 4.12 (ddd, 1H, J_gem = 10.4, J_H,P = 6.0, J_5′a,4′ = 3.6, H-5′a); 4.56 (dt, 1H, J_3′,2′ = 6.2, 3.6, J_3′,4′ = 3.6, H-3′); 6.27 (dd, 1H, J_1′,2′ = 7.1, 6.0, H-1′); 7.49 (dd, 1H, J_{5′′′,4′′′} = 7.3, J_{5′′′,6′′′} = 4.8, H-5′′′); 7.88 (d, 1H, J_{5′′,4′′} = 7.7, H-5′′); 7.99 (m, 2H, H-4′′,4′′′); 8.23 (d, 1H, J_{3′′,4′′} = 7.9, H-3′′); 8.27 (d, 1H, J_{3′′′,4′′′} = 7.9, H-3′′′); 8.45 (s, 1H, H-6); 8.70 (d, 1H, J_{6′′′,5′′′} = 4.8, H-6′′′); ¹³C NMR (150.9 MHz, CD₃OD): 41.66 (CH₂-2′); 65.10 (d, J_C,P = 4.5, CH₂-5′); 72.41 (CH-3′); 82.78 (bpy-C≡C); 87.88 (CH-1′); 88.36 (d, J_C,P = 8.4, CH-4′); 91.71 (C-5); 94.66 (bpy-C≡C); 122.26 (CH-3′′); 122.90 (CH-3′′′); 125.64 (CH-5′′′); 128.81 (CH-5′′); 138.95 (CH-4′′′); 139.44 (CH-4′′); 143.86 (C-6′′); 147.22 (CH-6); 150.57 (CH-6′′′); 156.50 (C-2′′′); 156.68 (C-2); 157.58 (C-2′′); 166.26 (C-4); ³¹P NMR (202.3 MHz, CD₃OD): 5.06; MS (ES⁻): found m/z: 484.2 (M), 485.2 (M+H), 486.2 (M+2H); HRMS (ES⁻): m/z calcd for C₂₁H₁₉O₇N₅P: 484.1028; found: 484.1028.

General procedure for Sonogashira cross-coupling – synthesis of modified dN^RTPs

Mixture CH₃CN/H₂O (1 : 2) (1.5 ml) and Et(i-Pr)₂N (10 equiv.) were added to an argon-purged flask containing halogenated nucleoside triphosphatedC^ITP or dA^ITP (60 mg), an alkyne 1a–c (1.5 equiv. for dC^ITP and 2 equiv. for dA^ITP) and CuI (10 mol%). In a separate flask, Pd(OAc)₂ (5 mol%) and TPPTS (5 equiv. to Pd) were combined, evacuated and purged with argon followed by addition of CH₃CN/H₂O (1 : 2) (0.5 ml). The mixture of catalyst was then injected into the reaction mixture and the reaction mixture was stirred at 80 °C for 1 h. The solvent was evaporated in vacuo. Products were purified by semi-preparative HPLC on C18 column using linear gradient of 0.1 M TEAB (triethylamonium bicarbonate) in H₂O to 0.1 M TEAB in H₂O/MeOH (1 : 1) as an eluent. Several co-distillations with water and conversion to sodium salt form (Dowex 50 in Na⁺ cycle) followed by freeze-drying from water, gave the products as white or yellow powder.

5-[(2′′,2′′′-bipyridin-6′′-yl)ethynyl]-2′-deoxycytidine 5′-O-triphosphate (dC^6bpyTP)

This compound was prepared according to the general procedure from 5-iodo-2′-deoxycytidine monophosphate dC^ITP and 1a in the yield 67%.

¹H NMR (499.8 MHz, D₂O, ref_dioxane = 3.75 ppm, pD = 7.1, phosphate buffer): 2.22 (dt, 1H, J_gem = 14.1, J_2′b,1′ = J_2′b,3′ = 6.8, H-2′b); 2.42 (ddd, 1H, J_gem = 14.1, J_2′a,1′ = 6.3, J_2′a,3′ = 4.1, H-2′a); 4.23 (m, 3H, H-4′,5′); 4.57 (dt, 1H, J_3′,2′ = 6.8, 4.1, J_3′,4′ = 4.1, H-3′); 6.11 (dd, 1H, J_1′,2′ = 6.8, 6.3, H-1′); 7.45 (bdd, 1H, J_{5′′′,4′′′} = 7.8, J_{5′′′,6′′′} = 4.2, H-5′′′); 7.57 (bd, 1H, J_{5′′,4′′} = 7.8, H-5′′); 7.92 (bt, 1H, J_{4′′,3′′} = J_{4′′,5′′} = 7.8, H-4′′); 7.95 (m, 2H, H-3′′, H-4′′′); 8.02 (bd, 1H, J_{3′′′,4′′′} = 7.8, H-3′′′); 8.03 (s, 1H, H-6); 8.54 (bd, 1H, J_{6′′′,5′′′} = 4.2, H-6′′′); ¹³C NMR (125.7 MHz, D₂O, ref_dioxane = 69.3 ppm, pD = 7.1, phosphate buffer): 41.93 (CH₂-2′); 67.92 (d, J_C,P = 5.6, CH₂-5′); 72.90 (CH-3′); 83.58 (bpy-C≡C); 88.16 (d, J_C,P = 8.7, CH-4′); 89.11 (CH-1′); 94.06 (C-5); 96.48 (bpy-C≡C); 124.25 (CH-3′′); 124.88 (CH-3′′′); 127.45 (CH-5′′′); 130.46 (CH-5′′); 141.35, 141.49 (CH-4′′, CH-4′′′); 144.23 (C-6′′); 148.11 (CH-6); 151.53 (CH-6′′′); 156.84 (C-2′′′); 158.03 (C-2′′); 158.21 (C-2); 167.00 (C-4); ³¹P NMR (202.3 MHz, D₂O, ref_{phosphate buffer} = 2.35 ppm, pD = 7.1): −21.06 (t, J = 19.7, P_β); −10.10 (d, J = 19.6, P_α); −6.74 (bd, J = 19.6, P_γ); MS (ES⁻): found m/z: 644.0 (M-1), 564.1 (M–PO₃H₂–1); HRMS (ES⁻): m/z calcd for C₂₁H₂₁O₁₃N₅P₃: 644.0354; found: 644.0348.

Primer extension, purification and analysis of the PEX products

Synthetic ONs were purchased from Sigma Aldrich (USA). Primer: 5′-CAT GGG CGG CAT GGG-3′; templates: 5′-CTA GCA TGA GCT CAG TCC CAT GCC GCC CAT G-3′(temp^rnd16), 5′-CCC GCC CAT GCC GCC CAT G-3′ (temp^C), 5′-CCCTCC CAT GCC GCC CAT G-3′ (temp^A), TCC CAT GCC GCC CAT G-3′ (temp^A1) (segments forming duplex with the primer are in italics, the replicated segments are in bold). Templates used in experiment involving the DBstv magnetoseparation procedure were biotinylated at their 5′ ends. Streptavidine magnetic beads were obtained from Sigma Aldrich (MagSelect, USA) or Novagen (MagPrep, USA), Pwo DNA polymerase from PeqLab (Germany), DyNAzyme II and Phusion DNA polymerases from Finnzymes (Finland), KOD XL DNA polymerase from Novagen, Vent (exo⁻), Deep Vent, Deep Vent (exo⁻) and Therminator DNA polymerases as well as T4 polynukleotide kinase and natural nucleoside triphosphate (dATP, dCTP, dGTP and dCTP) from New England Biolabs (Great Britain) and γ-³²P-ATP from Izotop, Institute of isotopes Co, Ltd. (Hungary).

Primer extension experiment for single incorporation by using Pwo polymerase. The reaction mixture (20 μl) contained Pwo polymerase (0.1 U/μl, 2 μl), dNTPs (either natural or modified, 4 mM, 1 μl), ³²P-prelabelled primer at 5′-end (3 μM, 1 μl) and template temp^A (3 μM, 1.5 μl) in 2 μl of corresponding buffer supplied by manufacturer. Reaction mixture was incubated for 30 min at 60 °C.

Primer extension experiment for single incorporation by using DyNAzyme II. The reaction mixture (20 μl) contained DyNAzyme II polymerase (0.1 U/μl, 1 μl), natural dNTPs (4 mM, 0.5 μl) and modified dN^RTPs (4 mM, 1 μl), ³²P-prelabelled primer at 5′-end (3 μM, 1 μl) and template temp^C (3 μM, 1.5 μl) in 2 μl of corresponding buffer supplied by manufacturer. Reaction mixture was incubated for 10 min at 60 °C.

Primer extension for multiple incorporation by using Pwo. The reaction mixture (20 μl) contained Pwo polymerase (0.1 U/μl, 2 μl), dNTPs (either natural or modified, 4 mM, 1 μl), ³²P-prelabelled primer at 5′-end (3 μM, 1 μl) and template temp^rnd16 (3 μM, 1.5 μl) in 2 μl of corresponding buffer supplied by manufacturer. Reaction mixture was incubated for 30 min at 60 °C.

Primer extension for multiple incorporation by using DyNAzyme II. The reaction mixture (20 μl) contained DyNAzyme II polymerase (1U/μl, 1 μl), natural dNTPs (4 mM, 1 μl) and modified dN^RTPs (4 mM, 2 μl), ³²P-prelabelled primer at 5′-end (3 μM, 1 μl) and template temp^rnd16 (3 μM, 1.5 μl) in 2 μl of corresponding buffer supplied by manufacturer. Reaction mixture was incubated for 30 min at 60 °C.

For magnetoseparation unlabelled primers and biotinylated templates were used.

Primer extension for kinetics study. The reaction mixture (20 μl) contained DNA polymerase: Pwo (0.1 U/μl, 2 μl), dATP/dA^RTP (4 mM, 1 μl), ³²P-prelabelled primer at 5′-end (3 μM, 1 μl) and template temp^A1 (3 μM, 1.5 μl) in 2 μl of corresponding buffer supplied by manufacturer. Reaction mixture was incubated at 60 °C for required time followed by stopping the reaction by addition of PAGE loading buffer and immediate heating.

Denaturating polyacrylamide gel electrophoresis. The products of the primer extension reaction were mixed with loading buffer (40 μl, 80% [w/v] formamide, 20 mM EDTA, 0.025% [w/v] bromphenole blue, 0.025% [w/v] xylene cyanol), heated 5 min at 95 °C and subjected to gel electrophoresis in 12.5% denaturating polyacrylamide gel containing 1xTBE buffer (pH 8) and 7% urea at 60 W for ∼60 min. Gel was dried and visualized by phosphoimager.

General procedure for complexation

Complexation of dN^RMPs. Complexes of modified nucleoside monophosphatesdN^RMPs with diverse transition metals were prepared by mixing 100 μl of aqueous solution corresponding monophosphate (100 μM) with 100 μl of divalent metal ions M²⁺ (50 μM, Cu(BF₄)₂·6H₂O, Ni(BF₄)₂·6H₂O, Zn(BF₄)₂·H₂O, Fe(BF₄)₂·6H₂O) at room temperature for 10 min.

Complexation of ON for recording UV-spectra. Double stranded ONs were prepared by PEX-experiment on larger scale. The reaction mixture (200 μl) contained Deep Vent polymerase (2 U/μl, 15 μl), dNTP (either natural or modified, 4 mM, 30 μl), unlabeled primer (100 μM, 12 μl), and temp^rnd16 (100 μM, 12 μl) in 20 μl of corresponding buffer supplied by manufacturer. Reaction mixture was incubated for 30 min. at 60 °C. PEX-products were purified by NucAway Spin Columns (Ambion), where 50 μl portions of each sample were applied on the top of the column. After collecting all the portions 0.5 equiv. of Fe(BF₄)₂·6H₂O to number of modification (0.24 μl, 10 mM) was added and the solution was mixed for 3 h (25 °C, 550 rpm).

Complexation of ON for gel electrophoresis. Double stranded ONs were prepared by PEX-experiment. The reaction mixture (20 μl) contained DNA polymerase: Pwo (0.1 U/μl, 2 μl), DyNAzyme II (0.1 U/μl, 1 μl), dNTP (either natural or modified, 4 mM, 1 μl), ³²P-prelabelled primer at 5′-end primer (3 μM, 1 μl), and temp (3 μM, 1.5 μl) in 2 μl of corresponding buffer supplied by manufacturer. Reaction mixture was incubated for 30 min. at 60 °C. For incorporation of dC^tpyTP using Dynazyme II polymerase and temp^C was used 0.5 μl of natural dNTPs (4 mM) and the mixture was incubated only for 15 min. After addition of 1 μl of Fe²⁺ (4 mM, Fe(BF₄)₂·6H₂O or FeCl₂) the solution was mixed for 3 h (25 °C, 550 rpm).

Non-denaturating SB polyacrylamide gel electrophoresis. The products of the primer extension reaction were mixed with loading buffer (4 μl, 40% [w/v] sacchrose, 0.2% [w/v] bromphenole blue, 0.2% [w/v] xylene cyanol) subjected to gel electrophoresis in 8% non-denaturating polyacrylamide gel containing 1xSB buffer (pH 8) and at 500 V for ∼3 h at room temperature. Gel was dried and visualized by phosphoimager.

Acknowledgements

This work is a part of the research projects Z4 055 0506 supported by the Academy of Sciences of the Czech Republic. It was specifically supported by the Czech Science Foundation (203/09/0317), by the Ministry of Education, Youth and Sports (LC512) and by Gilead Sciences, Inc. (Foster City, CA, U. S. A.).

Notes and references

1. (a) R. R. Breaker, Curr. Opin. Chem. Biol., 1997, 1, 26; (b) M. Famulok, G. Mayer and M. Blind, Acc. Chem. Res., 2000, 33, 591; (c) M. Famulok, Curr. Opin. Mol. Ther., 2005, 7, 137; (d) K. V. Gothelf and T. H. LaBean, Org. Biomol. Chem., 2005, 3, 4023; (e) F. A. Aldaye and H. F. Sleiman, Pure Appl. Chem., 2009, 81, 2157.
2. (a) G. H. Clever, C. Kaul and T. Carell, Angew. Chem., Int. Ed., 2001, 46, 6226; (b) C. Switzer, S. Sinha, P. H. Kim and B. D. Heuberger, Angew. Chem., Int. Ed., 2005, 44, 1529; (c) G. H. Clever, Y. Söltl, H. Burks, W. Spahl and T. Carell, Chem.–Eur. J., 2006, 12, 8708; (d) K. Tanaka, A. Tengeiji, T. Kato, N. Toyama, M. Shiro and M. Shionoya, J. Am. Chem. Soc., 2002, 124, 12494; (e) N. Zimmermann, E. Meggers and P. G. Schultz, J. Am. Chem. Soc., 2002, 124, 13684; (f) K. Tanaka and M. Shionoya, J. Org. Chem., 1999, 64, 5002; (g) Ch. Switzer and D. Shin, Chem. Commun., 2005, 1342; (h) H. Weizman and Y. Tor, J. Am. Chem. Soc., 2001, 123, 3375.
3. (a) A. A. Gorodetsky and J. K. Barton, Langmuir, 2006, 22, 7917; (b) P. K. Bhattacharya, H. J. Lawson and J. K. Barton, Inorg. Chem., 2003, 42, 8811; (c) C. Stinner, M. D. Wightman, S. O. Kelley, M. G. Hill and J. K. Barton, Inorg. Chem., 2001, 40, 5245; (d) J. L. Kisko and J. K. Barton, Inorg. Chem., 2000, 39, 4942; (e) R. E. Holmlin, J. A. Yao and J. K. Barton, Inorg. Chem., 1999, 38, 174; (f) S. J. Franklin, C. R. Treadway and J. K. Barton, Inorg. Chem., 1998, 37, 5198; (g) A. J. Boersma, R. P. Megens, B. L. Feringa and G. Roelfes, Chem. Soc. Rev., 2010, 39, 2083; (h) G. Roelfes and B. L. Feringa, Angew. Chem., Int. Ed., 2005, 44, 3230; (i) G. Roelfes, A. J. Boersma and B. L. Feringa, Chem. Commun., 2006, 635.
4. (a) M. Kalek, A. S. Madsen and J. Wengel, J. Am. Chem. Soc., 2007, 129, 9392; (b) M. M. Rodriguez-Ramos and J. J. Wilker, JBIC, J. Biol. Inorg. Chem., 2010, 15, 629; (c) S. Ghosh, I. Pignot-Paintrand, P. Dummy and E. Defrancq, Org. Biomol. Chem., 2009, 7, 2729; (d) G. Bianké, V Chaurin, M. Egorov, J. Lebreton, E. C. Constable, C. E. Housecroft and R. Häner, Bioconjugate Chem., 2006, 17, 1441; (e) K. Wiederholt and L. W. McLaughlin, Nucleic Acids Res., 1999, 27, 2487.
5. M. Vrábel, P. Horáková, H. Pivoňková, L. Kalachova, H. Černocká, H. Cahová, R. Pohl, P. Šebest, L. Havran, M. Hocek and M. Fojta, Chem.–Eur. J., 2009, 15, 1144.
6. (a) D. J. Hurley and Y. Tor, J. Am. Chem. Soc., 1998, 120, 2194; (b) D. J. Hurley and Y. Tor, J. Am. Chem. Soc., 2002, 124, 3749; (c) D. J. Hurley and Y. Tor, J. Am. Chem. Soc., 2002, 124, 13231; (d) H. Weizman and Y. Tor, J. Am. Chem. Soc., 2002, 124, 1568; (e) D. J. Hurley, S. E. Seeman, J. C. Mazura and Y. Tor, Org. Lett., 2002, 4, 2305; (f) S. I. Khan, A. E. Beilstein and M. W. Grinstaff, Inorg. Chem., 1999, 38, 418; (g) S. I. Khan, A. E. Beilstein, G. D. Smith, M. Sykora and M. W. Grinstaff, Inorg. Chem., 1999, 38, 2411; (h) J. Telser, K. A. Cruickshank, K. S. Schanze and T. L. Netzel, J. Am. Chem. Soc., 1989, 111, 7221.
7. (a) D. Mitra, N. Di Cesare and H. F. Sleiman, Angew. Chem., Int. Ed., 2004, 43, 5804; (b) H. Yang and H. F. Sleiman, Angew. Chem., Int. Ed., 2008, 47, 2443; (c) H. Yang, A. Z. Rys, C. K. McLaughlin and H. F. Sleiman, Angew. Chem., Int. Ed., 2009, 48, 9919; (d) H. Yang, C. K. McLaughlin, F. A. Adlaye, G. D. Hamblin, A. Z. Rys, I. Rouiller and H. F. Sleiman, Nat. Chem., 2009, 1, 390; (e) J. S. Choi, C. W. Kang, K. Jung, J. W. Yang, Y.-G. Kim and H. Han, J. Am. Chem. Soc., 2004, 126, 8606; (f) K. M. Stewart and L. W. McLaughlin, J. Am. Chem. Soc., 2004, 126, 2050; (g) K. W. Gothelf, A. Thomsen, M. Nielsen, E. Clóand and R. S. Brown, J. Am. Chem. Soc., 2004, 126, 1044. Review: (h) K. Tanaka and M. Shionoya, Coord. Chem. Rev., 2007, 251, 2732. Review: (i) H. Yang, K. L. Metera and H. F. Sleiman, Coord. Chem. Rev., 2010, 254, 2403. Review: (j) T. J. Bandy, A. Brewer, J. R. Burns, G. Marth, T. Nguyen and E. Stulz, Chem. Soc. Rev., 2011, 40, 138.
8. L. Kalachova, R. Pohl and M. Hocek, Synthesis, 2009, 105.
9. M. Vrábel, R. Pohl, I. Votruba, M. Sajadi, S. A. Kovalenko, N. P. Ernsting and M. Hocek, Org. Biomol. Chem., 2008, 6, 2852.
10. S. T. Gaballah, C. E. Kerr, B. E. Eaton and T. L. Netzel, Nucleosides, Nucleotides Nucleic Acids, 2002, 21, 547.
11. (a) T. Ruhl and E. Stulz, Supramol. Chem., 2010, 22, 103; (b) J. R. Burns, J. Zekonyte, G. Siligardi, R. Hussain and E. Stulz, Molecules, 2011, 16, 4912.
12. T. Ehrenschwender, A. Barth, H. Puchta and H.-A. Wagenknecht, Org. Biomol. Chem. 10.1039/c1ob06421e.
13. (a) H. Hashimoto, M. G. Nelson and C. Schwitzer, J. Am. Chem. Soc., 1993, 115, 7128; (b) H. Rosemeyer, N. Ramzaeva, E.-M. Becker, E. Feiling and F. Seela, Bioconjugate Chem., 2002, 13, 1274; (c) J. A. Brazier, T. Shibata, J. Townsley, B. F. Taylor, E. Frary, N. H. Williams and D. M. Williams, Nucleic Acids Res., 2005, 33, 1362.
14. Reviews: (a) S. H. Weisbrod and A. Marx, Chem. Commun., 2008, 5675; (b) M. Hocek and M. Fojta, Org. Biomol. Chem., 2008, 6, 2233.
15. (a) S. Jäger, G. Rasched, H. Kornreich-Leshem, M. Engesser, O. Thum and M. Famulok, J. Am. Chem. Soc., 2005, 127, 15071; (b) C. Lam, C. Hipolito and D. M. Perrin, Eur. J. Org. Chem., 2008, 4915; (c) T. Gourlain, A. Sidorov, N. Mignet, S. J. Thorpe, S. E. Lee, J. A. Grasby and D. M. Williams, Nucleic Acids Res., 2001, 29, 1898; (d) L. H. Thoresen, G.-S. Jiao, W. C. Haaland, M. L. Metzker and K. Burgess, Chem.–Eur. J., 2003, 9, 4603; (e) M. Kuwahara, J. Nagashima, M. Hasegawa, T. Tamura, R. Kitagata, K. Hanawa, S. Hososhima, T. Kasamatsu, H. Ozaki and H. Sawai, Nucleic Acids Res., 2006, 34, 5383; (f) A. Shoji, T. Hasegawa, M. Kuwahara, H. Ozaki and H. Sawai, Bioorg. Med. Chem. Lett., 2007, 17, 776; (g) S. Obeid, M. Yulikow, G. Jeschke and A. Marx, Angew. Chem., Int. Ed., 2008, 47, 6782; (h) B. Holzberger and A. Marx, Bioorg. Med. Chem., 2009, 17, 3653; (i) V. Borsenberger and S. Howorka, Nucleic Acids Res., 2009, 37, 1477; (j) V. Borsenberger, M. Kukwikila and S. Howorka, Org. Biomol. Chem., 2009, 7, 3826; (k) C. T. Wirges, J. Timper, M. Fischler, A. S. Sologubenko, J. Mayer, U. Simon and T. Carell, Angew. Chem., Int. Ed., 2009, 48, 219; (l) K. Gutsmiedl, D. Fazio and T. Carell, Chem.–Eur. J., 2010, 16, 6877; (m) A. Baccaro and A. Marx, Chem.–Eur. J., 2010, 16, 218; (n) N. Ramsay, A.-S. Jemth, A. Brown, N. Crampton, P. Dear and P. Holliger, J. Am. Chem. Soc., 2010, 132, 5096.
16. (a) P. Čapek, H. Cahová, R. Pohl, M. Hocek, C. Gloekner and A. Marx, Chem.–Eur. J., 2007, 13, 6196; (b) P. Brázdilová, M. Vrábel, R. Pohl, H. Pivoňková, L. Havran, M. Hocek and M. Fojta, Chem.–Eur. J., 2007, 13, 9527; (c) H. Cahová, L. Havran, P. Brázdilová, H. Pivoňková, R. Pohl, M. Fojta and M. Hocek, Angew. Chem., Int. Ed., 2008, 47, 2059; (d) H. Cahová, R. Pohl, L. Bednárová, K. Nováková, J. Cvačka and M. Hocek, Org. Biomol. Chem., 2008, 6, 3657; (e) H. Macíčková-Cahová and M. Hocek, Nucleic Acids Res., 2009, 37, 7612; (f) J. Riedl, P. Horáková, P. Šebest, R. Pohl, L. Havran, M. Fojta and M. Hocek, Eur. J. Org. Chem., 2009, 3519; (g) V. Raindlová, R. Pohl, M. Šanda and M. Hocek, Angew. Chem., Int. Ed., 2010, 49, 1064; (h) S. Ikonen, H. Macíčková-Cahová, R. Pohl, M. Šanda and M. Hocek, Org. Biomol. Chem., 2010, 8, 1194; (i) H. Macíčková-Cahová, R. Pohl and M. Hocek, ChemBioChem, 2011, 12, 431; (j) P. Kielkowski, R. Pohl and M. Hocek, J. Org. Chem., 2011, 76, 3457; (k) H. Macíčková-Cahová, R. Pohl, P. Horáková, L. Havran, J. Špaček, M. Fojta and M. Hocek, Chem.–Eur. J., 2011, 17, 5833.
17. P. S. Braterman, J. I. Song and R. D. Peacock, Inorg. Chem., 1992, 31, 555.
18. V. Grosshenny, F. M. Romero and R. Ziessel, J. Org. Chem., 1997, 62, 1491.
19. P. Kielkowski, H. Macíčková-Cahová, R. Pohl and M. Hocek, Angew. Chem., Int. Ed., 2011, 50, 8727.

---

Footnote

† Electronic supplementary information (ESI) available: Full experimental part and spectral data, additional PAGEs, MALDI and additional UV/vis spectra. See DOI: 10.1039/c1ob06359f

---